The newly discovered uncoupling proteins, UCP2 and UCP3, are thought to be involved in regulation of body weight, diabetes, and non-shivering thermogenesis. UCP2 and UCP3 are mitochondrial proteins. As indicated by their name, they are thought to be terminal energy dissipators in the energy balance cycle. However, nothing is known about their transport mechanisms or regulation. Accordingly, the first goal of this project is to determine their transport functions following gene expression in bacteria, protein isolation, and reconstitution into liposomes. We will determine what is transported by the proteins, and in particular, whether their transport mechanisms are consistent with uncoupling in vivo. The second goal is to characterize regulation of UCP2 and UCP3 by nucleotides and long-chain acyl CoA esters. We will investigate inhibition and binding of the family of nucleotides, together with the pH- and Mg2+-dependence of inhibition/binding. Preliminary results indicate that UCP1, UCP2 and UCP3 differ in their sensitivity to nucleotides. Accordingly, the third goal is to identify amino acids in the UCPs that are critical for regulation of nucleotide binding and inhibition, using site-directed mutagenesis and assays of function and binding. Finally, it is crucial to verify that native UCP2 and UCP3 are functionally expressed in the target tissues. Thus, the fourth goal is to identify and assay native UCP2 and UCP3 in mitochondria from brown fat, heart, kidney, skeletal muscle, white fat, and lymphocytes. The study of purified, reconstituted proteins in Goals 1-3 will employ ion-specific fluorescent probes to quantitate transport. The study of isolated mitochondria in Goal 4 will employ antibodies to verify the presence of proteins, and respiration and light scattering experiments to quantitate uncoupling and transport. The significance of this project lies in determining the biochemical function and regulation of UCP2 and UCP3, which are unknown.